Gram negative bacteria cause a wide variety of diseases in humans and animals. These include plague, caused by Yersinia pestis, typhoid fever, caused by Salmonella typhi, gonorrhea, caused by Neisseria gonorrhoeae, dysentery, caused by Shigella dysenteriae, gastroenteritis, commonly caused by Salmonella typhimurium, Escherichia coli, and Campylobacter jejuni, bacterial sepsis, caused primarily by Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, and septicemic diseases in cattle and pigs, caused by Salmonella dublin and Salmonella choleraesuis, respectively.
Humans and animals have evolved many defenses to infection by gram negative bacteria. One of the first lines of defense are the body's phagocytic cells. These cells engulf invading microorganisms and kill them by various methods, such as the release of proteolytic enzymes and oxygen radicals.
Unfortunately, many types of bacteria have evolved means to inhibit or resist the many microbicidal substances in phagocytic cells, thereby allowing them to survive within the cells. Such facultative intracellular pathogens are a clinically important group of bacteria. They include bacteria from the genera Salmonella, Yersinia, Shigella, and Neisseria.
Because the intraphagocytic environment is so hostile to bacteria, it seems reasonable to assume that the selection of bacteria from within phagocytes would follow the Darwinian principle of survival of the fittest. Current reports indicate that, in order to survive in phagocytes, Salmonellae must possess virulence attributes, such as plasmids, porins, and other outer membrane proteins related to virulence. See Taira, et al., Microbial Pathogenesis, 7:165-173 (1989), Tufano, et al., Microbial Pathogenesis, 7:337-346 (1989), and Gulig, Microbial Pathogenesis, 8:3-11 (1990), all of which are incorporated herein by reference. It has also been reported that mutants unable to survive in macrophages have lost immunogenicity and virulence when compared to their parental strains. See Buchmeier, et al., Infection and Immunity, 57:1-7 (1989), Fields, et al., Science, 243:1059-1062 (1989), and Buchmeier, et al., Science, 248:730-732 (1990), all of which are incorporated herein by reference. Therefore, the reasonable expectation would be that Salmonellae able to survive in phagocytic cells would possess optimal virulence properties.
Surprisingly, the inventor has discovered the opposite result to that expected. Salmonellae that survived serial passages through live phagocytic cells or their lysosomal products exhibited decreased virulence and, after a sufficient number of passages, became avirulent. The avirulent bacteria still produced an immunogenic response when innoculated into an animal host, thereby providing the basis for vaccines against gram negative bacteria.
Such vaccines would be highly desirable because such bacteria, particularly the facultative intracellular pathogens, are often able to evade the body's defense mechanisms. A vaccine would prepare and enhance the defense mechanisms prior to significant invasion by the bacteria against which the vaccine is directed. Live, avirulent bacteria, as opposed to killed bacteria or inactivated toxins, are particularly desirable as vaccines because they usually provide a broader immune system response.